Project Summary DNA and RNA aptamers are a useful class of synthetic affinity reagents. However, their performance can be greatly improved through the site-specific incorporation of chemically modified, ?non-natural? nucleotides that provide a greater chemical repertoire to enable superior aptamer affinity and specificity. Because a broad spectrum of chemical functional groups can be incorporated, non-natural aptamers offer the exciting potential for targeting molecules for which the generation of monoclonal antibodies remains difficult, such as small- molecule drugs, metabolites and carbohydrates. Unfortunately, the access to non-natural aptamers is severely limited. This is because the process of generating non-natural aptamers is technically challenging and limited to a few specialized laboratories. The goal of this project is to develop an integrated instrument, the Non-Natural Aptamer Array (N2A2) that eliminates these bottlenecks and enable rapid and facile non-natural aptamer discovery at virtually any research laboratory. The N2A2 will be built on a modified version of a benchtop commercial sequencer (Illumina MiSeq), and will perform every stage of non-natural aptamer discovery? including sequencing, screening and binding measurements?as part of a single work-flow. There are three main innovative aspects of our N2A2 system. First, our approach will entirely eliminate the need for polymerase engineering, and thus allows us to incorporate virtually any chemical functional group through click chemistry. Second, N2A2 will enable us to directly obtain the binding affinity (Kd) of ~10^7 aptamers directly in complex samples (e.g. cell lysate or serum), thereby resulting in aptamers with high-specificity. Finally, we will develop a machine-learning (ML) approach to identify key motifs (?k-mers?) and predict novel sequences with potentially higher affinity and specificity that can be tested using the N2A2 instrument. We believe this powerful combination of massively parallel, sequence-linked binding measurements with ML-based predictions will allow us to explore sequence space that is currently inaccessible to traditional in vitro selection methods, and enable us to discover aptamers with superior performance. The success of this project will produce an integrated instrument that greatly streamlines and accelerates the discovery of non-natural aptamers for a wide range of targets in complex media. The instrument is based on a commercially available sequencer and we will make all software available to the public. In this way, we believe the N2A2 instrument could broadly expand access to robust, high quality, custom affinity reagents for biomedical research and clinical diagnostics.